The invention relates to a model system for studying the progress of viral diseases and for assessing possible therapeutic and diagnostic protocols. Specifically, the invention concerns three-dimensional histocultures of adult lymph nodes and tonsils which provide substrates for infection, study, and discovering of new therapeutics and diagnostics.
The problem of providing a suitable model system for studying the progress of human immunodeficiency virus (HIV) and other viral diseases is well known. No in vivo model in animals for HIV, for example, is available at this time. Standard in vitro primary cell cultures do not mimic the full cellular repertoire within the tissues of an organism, and do not provide appropriate supracellular organization. Thus, it would be advantageous to have an in vitro model which more accurately mimics the behavior of the cells in the context of the organism.
Rosenszweig, M. et al., Leukemia (1994) 8:Suppl. 1, S163-S165 and Bonyhadi, M. L. et al., AIDS Res Hum Retrovir (1995) 11:1073-1080 have cultured neonatal thymus as a tissue model for HIV pathogenesis. However, while these cultures support productive infection of HIV-1, they show thymocyte cytopathology and profile changes only after infection with macrophage-tropic but not lymphocyte-tropic strains. The histocultures of the present invention can be derived from adult tissues and are thus more representative of the progress of infection.
Various methods for culturing tumor cells in three-dimensional culture are known. Some of these are summarized in Leighton, J. et al., Cancer Res (1957) 17:929-941. A system for culturing human tumors in vitro in three dimensions was described by Freeman, A. E. and Hoffman, R. M., Proc Natl Acad Sci USA (1986) 83:2694-2698. Furthermore, a three-dimensional skin culture which could be used to evaluate toxicity and the effect of compounds on hair growth was described in WO92/15700 published Sep. 17, 1992.
Thus, applying techniques for three-dimensional histoculture, a model system for viral infection, particularly HIV infection, has been provided by the present invention.
In addition to providing a model system for viral infection, the histoculture techniques of the present invention are useful as model systems to study antigen-specific immunoregulation in normal lymphoid tissue. Unlike previously described systems, the antigens can be applied directly.
Prior art systems for study of immunization of mouse or human lymphocyte suspensions with recall antigen in vitro require careful attention to cell density, culture vessel geometry, agitation, oxygenation and antigen concentration. These systems are described by Mishell, R. I., et al., Science (1968) 153:1004-1006; Hoffman, M. K., et al., Nature (1973) 243:408-410; Lana, H. C., et al., J Exp Med (1981) 154:1043-1057.
The invention is directed to a histoculture system wherein lymph node or tonsil tissue is supported in a three-dimensional, structurally faithful system to serve as a model for viral infection, particularly HIV infection, as well as a model for response to antigen.
Thus, in one aspect, the invention is directed to a histoculture system which is useful as an in vitro model for viral infection or for response to an antigen which system comprises a flexible macromolecular, porous matrix, and supported thereon, an integral macroscopic section of animal lymph node tissue or tonsil tissue, said matrix immersed in a suitable culture medium wherein the surface of the medium is approximately congruent with the interface between the tissue and the matrix. The section of tissue is then infected with an amount of virus effective to maintain growth of the virus or is stimulated with an antigen.
In other aspects, the invention is directed to the in vitro histoculture system which is thus infected with a virus. In still other aspects, the invention is directed to a method to chart the progress of viral infection using the histoculture system of the invention and to methods to identify therapeutic compounds and protocols effective against the infection using the histoculture as a model, or as a diagnostic over the course of treatment administered to a subject.
In still another aspect, the invention is directed to a method to chart the progress of an immunologic response to a specific antigen.
As the histoculture system of this invention can mount an immune response, as well as support infection by a virus, e.g., HIV, the study of the progress of infection is enhanced by tracking the ability of the infection to inhibit the immune response, causing immunodeficiency in vitro.